Diagnostic Test for Infectious Diseases in Cattle

ABSTRACT

The invention relates to an isolated or recombinant protein from the shark  Heterodontus francisci , which has bovine-erythrocyte-recognition activity and which can bind to sequences of antigens and/or proteins that are characteristic of infectious diseases. Once the aforementioned protein is bound to specific antigens of infectious diseases, it can haemagglutinate upon recognizing the bovine erythrocytes and antibodies characteristic of said diseases, which are present in the active state in biological samples such as whole blood, plasma or serum of bovine origin. The invention also relates to methods for protecting the detection of antibodies characteristic of infectious diseases, using the purified periplasmic extract or fusion protein, optionally purifying the recombinant protein.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the technical field of veterinary andbiotechnology, more specifically it relates to the detection ofantibodies typical of infectious diseases through a reaction ofhemagglutination; since it provides an isolated or recombinant proteinderived from shark Heterodontus francisci, a fusion protein containingsaid protein and a method for detecting antibodies to infectiousdiseases.

BACKGROUND

To date there are a lot of drugs and vaccines for various infectiousdiseases of cattle, but in some cases are not as effective if thedisease is in an advanced stage. Millions of dollars are spent onresearch, which is mostly aimed at finding an effective treatment,downplaying the timely detection of the disease through a good method,which in many cases can lead to appropriate treatment with excellentresults and offer a control on these diseases (1).

Parallel to that investment, statistics showing areas in which diseasessuch as bovine tuberculosis and brucellosis have not been eradicatedhave not decreased significantly, representing a major public healthproblem because they are considered zoonotic diseases (OIE, 2004)(NOM-031-ZOO-1995). (2,3)

In Mexico, the 83.11% of the country is in the process of eradication ofbovine tuberculosis with a prevalence less than 0.50%, the rest of thecountry has a prevalence of less than 2.05%. As for brucellosis, it hasbeen eradicated only in the state of Sonora, but in the rest of thecountry it has not been possible (SENASICA, 2011). Upon controlling anderadicating bovine tuberculosis a potential source for human infectionwill be eliminated and this has been demonstrated in several countriesthrough campaigns of prevention, control and eradication of tuberculosis(NOM-031-ZOO-1995).

Each year, hundreds of millions of animals are slaughtered in the worldbecause they are infected by these pathogens, causing great economiclosses to livestock (NOM-031-ZOO-1995). The existing problem for both isthe lack of early detection, which would allow a best control over thedisease. This lack of detection in the case of brucellosis is because anunequivocal diagnosis can only be made by isolation and identificationof Brucella, but in situations where bacteriological analysis is notpossible, the diagnosis can be based on serological methods. There is nosingle test that allows to identify the disease, and normally acombination of growth characteristics and bacteriological andserological methods is required to determine whether the animal isinfected (OIE, 2004). In the case of bovine tuberculosis, there is noclinical evidence of the disease in cattle until they have developedvery extensive lesions. The measures implemented by the government arereflected in the rigorous application of the tuberculin test and theselection of cattle with positive reaction, measures that have not beenvery effective to date, so in general have been unsuccessful (OIE,2004).

Moreover, other factors also influence the non timely detection ofinfectious diseases in cattle, such as the level of development, thequality of health systems, and access to accurate and cost-effectivemeans of diagnosis in each country. The majority of developingcountries, which lead the statistics of these diseases, have thisproblem. However, although some countries with economic potential havenew technologies, including some commercially available for detection ofthe disease, they are expensive and inaccessible for most of livestockfarmers. Also, diagnosis is usually reached when the damage isirreversible (OIE, 2004). Therefore, the need to create products thatare sensitive to detect diseases even without clinical signs and at anystage of the disease is a priority. In that sense, the diagnosticmethods used until today can be effective, but require careful samplepreparation, trained staff, and especially are time consuming. Othermethods that can be considered fast are not very accurate and theresults are not reliable.

The classic test to detect bovine tuberculosis is the tuberculin, one ofthe problems of this test is that it does not detect animals withoutdelayed-type hypersensitivity (DTH) to Mycobacterium bovis, termedanergic, either with a disseminated infection or a recent infection,because in such cases the cellular immunity is depressed or indevelopment (Ritacco, 1991). On the other hand, they are laboratorytests of blood, such as lymphocyte proliferation test, the interferongamma and enzyme immunoassay. The logistics and performing of thesetests in the laboratory may be a limiting factor, thus more comparativestudies of these new tests and skin tests in different field conditionsare needed (OIE, 2004). Another disadvantage of tuberculin is that has ahigh number of false positives, because it is used a protein extractthat induces cross-reactivity with non-pathogenic mycobacteria.

Works published such as Patarroyo et al. support the use ofMycobacterium antigens like ESAT-6 for tuberculosis detection, as it hasbeen shown that this antigen can detect active or latent Mycobacterium.In this sense, the document US20060024332A1, teaches the use of a fusionprotein, consisting of recombinant antigens like ESAT-6 to stimulateleucocytes and producing gamma interferon. It is considered a specificand sensitive method, but in economic terms the cost is very high andimpractical. Other published works, such as US20100166786A1,US20100015096A1, US006982085B2 and US7261897B2 show the use of fusionproteins, consisting of at least two antigens of tuberculosis, andalthough they have the function of detecting tuberculosis, its mainobjective is directed to be used as preventive vaccines and also requirespecialized equipment and that raises the cost of the product, besidesbeing directed to humans so far.

The detection of brucellosis is made by tests with buffered Brucellaantigen (rose bengal) and the buffered plate agglutination test, as wellas complement fixation test, enzyme-linked immunosorbent assay (ELISA)or fluorescence polarization assay. Despite they can be used to screenherds and individual animals, is a fact that no single serological testis suitable for each and every one of the epidemiological situations.Therefore, the reactivity of the samples that are positive in screeningtests should be confirmed using a confirmatory strategy established. Theindirect ELISA or the milk ring test, made with whole milk samples, areeffective for analyzing and controlling brucellosis in dairy cows, butthe milk ring test is less reliable in large herds. Anotherimmunological test is the Brucellin skin test, which is used in analysisor as a confirmatory test in positive herds (OIE, 2004).

To a large extent several diagnostic tests for tuberculosis andbrucellosis have been disclosed, which characterize by possessingantigens characteristic of the disease, or antigens that may be mergedwith another protein that can detect the disease, despite they mainlyare used as vaccines. Although some have high sensitivity andspecificity according to the investigations reported, US7632646B1 orUS20100166786A1 have the disadvantages of being uneconomical,impractical and most can hardly be used in non-urban areas, causing alarge consumption of transportation time, equipment and personneltraining.

In order to provide a product that suppresses these drawbacks also havebeen created diagnostic tests that involve fusion or recombinantproteins, which are characterized by having a protein that recognizesproteins of bovine erythrocytes and other protein that detects antigensor antibodies characteristic of an infectious disease, producing ahemagglutination reaction (Shohet et al. 1985). In this sense, for sometime now are available works such as WO9324630 and US7585508B1 that areclear examples of this type of recombinant proteins used in erythrocyteprotein recognition by one of its proteins. In the first study, amalaria peptide is identified, and in the second a monoclonal antibodyfrom a hybridoma; the latter shows that by merging with disease-specificantigens they produce a hemagglutination reaction, with a sensitivity of100%. However, the use of larger volumes and concentrations is mentionedfor recognizing erythrocyte protein than the protein that is the objectof this invention, which has a sensitivity in the range of 90-100%, witha single addition of protein, which makes it more convenient, fast andefficient than previous ones.

This invention proposes a protein from shark Heterodontus francisci,which recognizes bovine erythrocyte membrane proteins and furthermorecan be fused with proteins such as ESAT-6, and infectious diseases, suchas tuberculosis and brucellosis. The recombinant protein can detect thedisease, recognizing bovine erythrocytes and antibodies produced by thedisease through a hemagglutination reaction.

Therefore, the invention disclosed herein is a good alternative todetect antibodies that may be present in infectious diseases oflivestock, which will make the process very fast and that can bedetected in cattle that present no symptoms and with infection in activestate. Another advantage of this invention to be protected is that thecosts of training personnel and logistical issues can be eliminated.Also is not limited to required concentrations of the bacillus to get aresult, what happens to other diagnostic tests that are also impracticalin rural areas.

Object of the Invention

The present invention aims to provide a recombinant protein fromHeterodontus francisci shark, which recognizes bovine erythrocytes andfurther may be fused with antigens related to infectious diseases. Theinvention also encompasses the protein extract for induction ofanti-bovine erythrocyte protein.

Another object of the invention is to provide a nucleic acid fragmentencoding the anti-bovine erythrocyte protein of Heterodontus franciscishark with the amino acid sequence SEQ. ID. No. 2, wherein at least 85%of its sequence must be the same as the SEQ. ID. No. 1, to recognizebovine erythrocytes.

The invention also aims to provide a fusion protein to detect antibodiescharacteristic of infectious diseases, wherein said protein comprisesthe anti-bovine erythrocyte protein from Heterodontus francisci, as wellas an antigen characteristic of an infectious disease.

Moreover, the invention provides a method for detecting antibodiescharacteristic of infectious diseases such as bovine tuberculosis andbrucellosis, which comprises applying an effective amount of the fusionprotein to a biological sample, wherein the result is observed through ahemagglutination reaction.

DISCLOSURE OF THE INVENTION

The characteristic details of the present invention are clearly shown inthe following description and accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the profile of the gene encoding both anti-bovineerythrocyte protein and ESAT-6 through an agarose gel of 2%, with anapproximate size of 300 bp for the gene coding for ESAT-6, located inlane 2, while in lane 3 is shown the gene encoding the anti-bovineerythrocyte protein with an approximate size of 350 bp, and in lane 1shows the 3100 bp ladder (PROMEGA).

FIG. 2 shows the profile of the anti-bovine erythrocyte-ESAT-6 fusionprotein through a 2% agarose gel, with an approximate size of 650 bplocated in lane 2, while lane 1 shows the 100 bp ladder (PROMEGA).

Sequence list SEQ. ID No: 1 shows the nucleotide sequence of the geneencoding the protein of anti-bovine erythrocyte derived from francisciHeterodontus identified as SEQ. ID. No. 1.

Sequence list SEQ. ID No: 2 shows the amino acid sequence of theanti-bovine erythrocyte protein derived from Heterodontus francisciidentified as SEQ. ID. No. 2.

Obtaining Fraction of Anti-Bovine Erythrocyte Protein from Heterodontusfrancisci Shark.

A non-immune library from Heterodontus francisci shark, which waspreviously developed at the Laboratory of Immunology and Toxins CICESEwas used for obtaining the fragment of anti-bovine erythrocyte protein.

The non-immune library was cloned using the pComb3X vector, and itsdisplay, selection and expression in soluble form. By means of the phagedisplay technique (Barbas, 2001), in round 4 of the non-immune library,a protein that is recognized by bovine erythrocytes was isolated. Withphages obtained in that round the strain ER2537 was infected at opticaldensity of 1 using SB medium with carbenicillin, to sow the sample in LBagar plates with carbenicillin al 100 μg/mL. Plates were incubatedovernight at 37° C. From the colonies obtained, single colonies wereselected to make a PCR with specific primers and amplify the geneencoding the anti-bovine erythrocyte protein under the followingconditions: one cycle of 5 min at 94° C., 30 cycles of 45 s at 56° C.,30 cycles of 45 s at 72° C. and 1 cycle of 10 min at 72° C. Fromamplified products, the positive clone number 11, which had the genefragment encoding the anti-bovine erythrocyte protein, SEQ. ID. No. 1,was selected to obtain the plasmid, following the directions of theQiagen miniprep kit (QIAGEN) which was sent to sequencing.

Expression of the Anti-Bovine Erythrocytes Protein from Heterodontusfrancisci.

Electroporation.

Once isolated the plasmid, 5 μL were incubated on ice for 1 min, then 50μL of electrocompetent BL21 (DE3) cells were added, and incubated on icefor 1 min. Subsequently, the mixture was poured into a cuvette forcarrying out the electroporation, following the conditions 2.5 kv, 200Ωfor 4 ms. Cuvette was washed with 1 mL of SOC medium, followed by twowashes with 2 mL of SOC medium at a temperature range of 24 to 30° C.,and was incubated for 1 hr at 37° C. at 250 rpm. 10 μL of that productof electroporation were seeded on LB plates with 100 g/mL ofcarbenicillin in order to isolate clones.

Induction and Protein Extraction of Anti-Bovine Erythrocytes from H.Francisci.

One of the clones isolated in the previous section was used to inducesecretion of the anti-bovine erythrocyte protein inoculating it in LBmedium containing 100 μg/L of carbenicillin and adding IPTG (1 mM),incubating for 5 hours at 37° C. Subsequently, the culture wascentrifuged at 4,000×g for 20 min, and the cell packet was resuspendedin 5 mL of Solution 1 (30 mM Tris-Cl, 20% sucrose, pH 8.0) and EDTA wasadded dropwise to a final concentration LMM and incubated in ice withstirring for 10 min. Afterwards it was centrifuged at 8,000×g for 20min, the supernatant was recovered and the pellet was resuspended in 5mL of Solution 2 (MgSO₄ 5 mM), which was incubated on ice with stirringfor 20 min and was centrifuged again at 8,000×g for 20 min, and thesupernatant was recovered. The protein present in the supernatantobtained by using Solution 2 for enzyme-linked immunoassay (ELISA) wasused to observe its expression.

Hemagglutination Assays.

To demonstrate that the anti-bovine erythrocyte protein exhibited ahemagglutination reaction being in contact with bovine erythrocytes, invitro assays were performed on culture plates with blood samples fromcattle.

Prior to conducting the tests, a sample of 5 mL of bovine peripheralblood was obtained via venipuncture. Serum sample is separated fromcellular package and the package is washed 3 times with 1×PBS solutionand resuspended in 10 mL of the same solution to use it later as asolution of washed erythrocytes. In the tests induction extract purifiedwas used and quantified to a concentration of 10 μg/mL.

The test consisted of adding to each well in triplicate, 30 μL of washederythrocytes solution; then was added the extract of anti-bovineerythrocyte protein in a volume range of 10-80 μL, allowing to stand for30 min. Alfalfa sprouts were used as positive control, and PBS1× asnegative control. Table 1 shows the effect of isolated protein fromHeterodontus francisci on bovine erythrocytes, where can be seen thatthe effect of hemagglutination is positive, which represents that theinvention can be used with effective results.

TABLE 1 Recognition of bovine erythrocytes by the anti-bovineerythrocyte protein in different amounts through a reaction ofhemagglutination. Sample 10 μL 20 μL 30 μL 40 μL 60 μL 80 μL Bovine ++++ +++ +++ +++ +++ erythrocytes Agglutination: + No agglutination: −

Obtaining Overlapping Anti-Bovine Erythrocyte Protein˜Antigen.

From the results shown above, the anti-bovine erythrocyte proteinisolated from H. francisci shark demonstrates its great ability torecognize erythrocytes through a hemagglutination reaction. Thus, byfusing the anti-bovine erythrocyte protein with antigens from bacteria,viruses or fungi which may cause diseases such as tuberculosis,brucellosis, rabies, among others, the pathology can be detected by thehemagglutination reaction through the recognition of erythrocytes andalso the antibodies to the disease, considering this invention so far,fast, effective and practical compared to those currently used.

For Detection of Bovine Tuberculosis, Anti-Bovine Erythrocytes—eSAT-6.

To detect bovine tuberculosis, ESAT-6 antigen was used. The gene of bothproteins, bovine erythrocytes and anti-ESAT-6 is used.

We proceeded to overlap them using a PCR reaction with ramp, under thefollowing conditions: 1 cycle of 5 min at 94° C., 5 cycles of 45 s at94° C., 5 cycles of 16.5 min starting with 50° C. and an increase of 1°C. every 45 s, followed by 25 cycles of 12.75 min at 55° C. with anincrease of 1° C. every 45 s, 1 cycle of 45 s at 72° C. and finally 1cycle of 10 min at 72° C. With the chimeric gene of anti-bovineerythrocytes and ESAT-6, it was digested with Sfil enzyme like thepComb3X vector, so that sticky ends were ligated, precipitating theproduct of digestion with 1 μL glycogen, 20 μL acetate 3M sodium and 70%ethanol, incubating for 2 hrs at −70° C. The mixture was centrifuged at16,000×g for 30 minutes, the pellet was resuspended in 20 μL ofnuclease-free water and quantitated for subsequent ligation with T4ligase enzyme (Promega). Then it is proceeded to electroporate intoelectrocompetent BL21 (DE3) cells and the product is seeded in LB agarplates with carbenicillin 100 μg/mL. Secretion of the protein is inducedin positive clone by adding IPTG (1 mM Promega) and incubating for 5hours at 37° C. The protein extraction was performed as described in“Induction, protein extraction and anti-bovine erythrocytes from H.francisci”. The ability to express the anti-bovine erythrocyte-ESAT-6fusion protein is measured by reading an enzyme-linked immunoassaycomprising: Plate the protein in triplicate and incubating for one hourat 37° C. Discard the content and block wells with BSA3%-PBSI,incubating for one hour at 37° C. The wells are washed with 0.05%Tween-PBS1× three times, added 50 μL of mouse monoclonal antibodyanti-HA (ROCHE) diluted 1:1000 in 1% BSA-PBS1× incubating 1 hr at 37° C.It is washed with 0.05% Tween-PBS1× three times and 50 μL of peroxidasesubstrate are added, consisting of 20 μg/mL ABTS, 1× citrate buffer,0.3% hydrogen peroxide, allowing to stand for 20 min without lightstimulus. In FIG. 2, the profile presented by overlapping of suchanti-bovine erythrocyte-ESAT-6 fusion protein is shown.

Hemagglutination Assays with Fusion Protein.

In order to demonstrate that the fusion protein (anti-bovine erythrocyteprotein-antigen) has the same ability to hemagglutinate bovine than thebovine erythrocyte protein unfused by recognizing antibodies toinfectious diseases, hemagglutination tests were performed with thefusion protein. Periplasmic extract of the fusion protein was used.

The procedure was the same as the hemagglutination assays outlined abovefor anti-erythrocyte protein, with the only difference that they hadcontacted an experimental sample. The only variations in methodologywere in the case of whole blood.

EXAMPLE 1 Effect of Anti-Bovine Erythrocyte-ESAT-6 Fusion Protein inBovine Serum Samples

To verify the functionality of the anti-bovine erythrocyte-ESAT-6 fusionprotein, samples of cattle with tuberculosis confirmed by otherconventional methods, like the acid fast bacilli (AFB) and culture (goldstandard), which even when shown to be reliable and safe tests, fordiagnosis of tuberculosis, involve undue cost and time compared to thisinvention. The samples were subjected to the following methodology: weretaken 25 μL total bovine blood, which was centrifuged at 750×g for 3 minand the cell package was washed with PBS1×. After completing the washingprocess, again the erythrocytes were centrifuged, the supernatant wasdiscarded and the erythrocytes were resuspended in 20 μL of experimentalserum. 25 μL of anti-bovine erythrocyte-ESAT 6 fusion protein was added,and allowed to incubate at room temperature for 30 min. Alfalfa sproutswere used as a positive control, and as negative control PBS1×.

Table 2 shows that in the samples occurred hemagglutination with aneffectiveness of 100% for the fusion protein, the same as a conventionalmethod, however, the advantage of using the anti-bovineerythrocyte-ESAT-6 fusion protein is observed in the ability to detectthe antibodies produced by the tuberculosis effectively and quickly,representing savings in time, expenses, supplies and infrastructure forthe health sector.

TABLE 2 Percentage of sensitivity of samples with TB confirmedexhibiting hemagglutination using conventional methods such as AFB andculture, and using antiglycophorin-ESAT-6 purified fusion protein. AFBand culture Anti-bovine erythrocytes - Samples Conventional method ESATfusion protein Bovine sera 100% 100%

By the above result, it can be implemented as a new technique to detectinfectious diseases such as tuberculosis, as anti-erythrocyte-ESAT-6fusion protein can detect antibodies in active or latent form by ahemagglutination reaction.

EXAMPLE 2 Effect of Anti-Bovine Erythrocyte-ESAT-6 Fusion Protein inWhole Blood from Cattle

The anti-bovine erythrocyte-ESAT-6 fusion protein not only has theability to be used on infected bovine serum samples, but can also beused on whole blood samples and have the same effect and sensitivity.This saves even more time and a quantity as large of sample is notrequired, as only uses 3 to 5 drops of blood. Drops of blood are dilutedin PBS 1×. In a watch glass 500 μL of the sample and 10 μL of the fusionprotein are placed with stirring, and after 15 minutes the result isinterpreted. Table 3 shows that 30 samples with tuberculosis confirmedpreviously by a conventional method have a sensitivity of 100% fortuberculosis antibody detection when antiglycophorin-ESAT-6 purifiedfusion protein is used.

TABLE 3 Percentage of sensitivity of whole blood samples, withtuberculosis confirmed by a conventional method and with theantiglycophorin-ESAT-6 purified protein. AFB and culture AntiglycophorinESAT-6 Samples Conventional method purified fusion protein 30 wholeblood 100% 100%

Given the above, the anti-bovine erythrocyte protein from Heterodontusfrancisci shark is novel because it has the property of recognizingerythrocytes of cattle in samples of whole blood or serum, and also hasthe ability to fuse with infectious diseases antigens, recognizingantibodies of the same via a hemagglutination reaction.

REFERENCES

-   1. Field Evaluation of the protective efficacy of Mycobacterium    bovis BCG vaccine against bovine tuberculosis. C. López Valencia, T.    Rentería Evangelista, J. de Jesús Williams, A. Licea Navarro, A. De    la Mora Valle, G. Medina Basaltos.-   2. Epidemiología molecular de las tuberculosis bovina y humana en    una zona endémica de Querétaro, Mexico. (Molecular epidemiology of    bovine and human tuberculosis in an endemic area of Querétaro,    Mexico). MC Laura Pérez-Guerrero, MVZ MSc, PhD Feliciano    Millán-Suazo, Q, MSc, PhD Camila Arriaga-Díaz, MVZ Cecilia    Romero-Torres, MC Minerva Escartín-Chávez. Approved on Jan. 17,    2008.-   3. Aislamiento e identificación de Mycobacterium bovis a partir de    muestras de expectoración de pacientes humanos con pacienets con    problemas de respiración crónicos. (Isolation and Identification of    Mycobacterium bovis from sputum samples from human patients with    patients with chronic breathing problems). Paola Toledo Ordó{umlaut    over (n)}ez, Feliciano Milian Sauzo, Marco Antonio Santillán Flores,    Isaura Carolina Ramírez Caslla. Accepted on Feb. 22, 1999.-   4. Reciprocal cellular and humoral immune Response in bovine    tuberculosis. RITACCA V, Lopez B, Cantor I N, Barrera I, Errico F,    Nader. Res. Vet. Sci. 1991; 50: 365-367.-   5. Mexican Official Standard NOM-031-ZOO-1995, Ministry of    Agriculture, Livestock and Rural Development, National Campaign    against Bovine Tuberculosis (Mycobacterium bovis).-   6. OIE-World Organization for Animal Health, 2004.-   7. Sagarpa, SENASICA, 2011.

1. A nucleic acid fragment obtained from shark Heterodontus francisci,comprising the sequence SEQ ID NO:
 1. 2. The fragment of claim 1,wherein the fragment encodes a protein with sequence SEQ ID NO:
 2. 3.The fragment of claim 2, wherein the protein recognizes bovineerythrocytes.
 4. The fragment of claim 2, wherein the fragment allowsthe fusion of the gene and the protein with sequences of infectiousdisease antigens.
 5. An isolated protein from Heterodontus franciscishark, wherein the isolated protein contains the sequence SEQ ID NO: 2.6. The isolated protein of claim 5, wherein the isolated proteinrecognizes bovine erythrocytes.
 7. The isolated protein of claim 6,wherein at least 85% of the nucleotide sequence of the isolated proteinis the same as the sequence SEQ ID NO: 1 to recognize bovineerythrocytes.
 8. The isolated protein of claim 5, wherein the fusionprotein can be fused with infectious diseases antigens.
 9. A method forobtaining a recombinant protein from Heterodontus francisci shark,comprising the following steps: a) obtaining a (non-immune) library ofHeterodontus francisci shark antibodies; b) cloning the library inEscherichia coli, c) selecting an anti-erythrocyte protein using phagedisplay technique, with: i) the sequence SEQ ID NO: 2; and ii) asequence having at least 85% similarity in codons of SEQ ID NO: 1; d)extracting the corresponding plasmid and cloning it into an expressionsystem, e) inducing the production of a recombinant protein, f)extracting the recombinant protein by osmotic shock, g) purifying therecombinant protein, and h) verifying the ability of the recombinantprotein to agglutinate bovine erythrocytes.
 10. A fusion protein, inwhich a domain comes from shark Heterodontus francisci, comprising: a) aprotein comprising the sequence SEQ ID NO: 2; and b) an infectiousdisease antigen.
 11. A fusion protein, in which a domain comes fromshark Heterodontus francisci, comprising: a) a protein whose sequencehas at least 85% identity with SEQ ID NO: 2; and b) an infectiousdisease antigen.
 12. The fusion protein of claim 11, wherein the fusionprotein recognizes bovine erythrocytes.
 13. The fusion protein of claim11, wherein the fusion protein has antibody recognition of infectiousdiseases.
 14. A method for obtaining a fusion protein in which a domaincomes from shark Heterodontus francisci, comprising the steps of: a)binding SEQ ID NO: 1 with a gene coding for an infectious diseaseantigen; b) cloning the overlap in an expression vector; c) cloning theexpression vector in an expression system; d) inducing the production ofa recombinant protein; e) extracting the recombinant protein; and f)purifying the recombinant protein.
 15. A composition comprising thefusion protein of claim 10 and a saline solution.
 16. An in vitro methodfor detecting antibodies to infectious diseases comprising the steps of:a) contacting a biological sample with a fusion protein from sharkHeterodontus francisci having: i) a protein having the amino acidsequence SEQ ID NO: 2; whose nucleotide sequence has at least 85%identity with SEQ ID NO: 1; and ii) an infectious disease antigen; andb) detecting the presence of antibodies associated with infectiousdiseases in the biological sample, using bovine erythrocyteagglutination.
 17. The method according to claim 16, wherein thebiological sample is derived from a subject under study.
 18. The methodaccording to claim 17, wherein the subject under study is a mammaliananimal.
 19. The method according to claim 18, wherein the mammaliananimal is a bovine.
 20. The method according to claim 16, wherein thebiological sample comprises whole blood, plasma or serum derived fromblood.
 21. (canceled)